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WO2008142198A2 - Procédé pour inhiber ou stimuler l'angiogenèse dans un sujet - Google Patents

Procédé pour inhiber ou stimuler l'angiogenèse dans un sujet Download PDF

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Publication number
WO2008142198A2
WO2008142198A2 PCT/FI2008/050262 FI2008050262W WO2008142198A2 WO 2008142198 A2 WO2008142198 A2 WO 2008142198A2 FI 2008050262 W FI2008050262 W FI 2008050262W WO 2008142198 A2 WO2008142198 A2 WO 2008142198A2
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WO
WIPO (PCT)
Prior art keywords
tcptp
peptide
seq
vegfr2
rplkkkmek
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PCT/FI2008/050262
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English (en)
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WO2008142198A3 (fr
Inventor
Johanna Ivaska
Elina Mattila
Original Assignee
Valtion Teknillinen Tutkimuskeskus
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Application filed by Valtion Teknillinen Tutkimuskeskus filed Critical Valtion Teknillinen Tutkimuskeskus
Priority to US12/601,108 priority Critical patent/US20100160228A1/en
Publication of WO2008142198A2 publication Critical patent/WO2008142198A2/fr
Publication of WO2008142198A3 publication Critical patent/WO2008142198A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to a method for inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signalling in an individual by administering an effective amount of an agent capable of activating T cell protein tyrosine phosphatase (TCPTP), and for inhibiting angiogenesis and treating or preventing diseases related thereto. Furthermore, this invention concerns a method for stimulating angiogenesis or for treatment or prevention of a disease benefiting from increasing the growth of blood vessels in an individual by inhibiting TCPTP.
  • VEGFR2 vascular endothelial growth factor receptor 2
  • the invention concerns also a novel peptide and a fusion protein based thereon.
  • VEGF vascular endothelial growth factor
  • VEGFRl high-affinity tyrosine kinase receptors
  • VEGFR2 appears to be the main mediator of VEGF-induced endothelial proliferation, survival, migration, tubular morphogenesis, and sprouting in adults (2).
  • the effects of VEGF are mainly controlled by its availability and by the activity of VEGFR signalling.
  • VEGFR2 signalling is equally important in limiting the response of VEGF in target cells.
  • inhibition of neovascularization is a promising new way of treating diseases such as cancer, certain retinopathies and other angiogenetic diseases.
  • Integrin-mediated adhesion to the extracellular matrix provides permissive signals to cells by supporting migration, cell survival, and signalling by receptor tyrosine kinases (RTKs) (4).
  • RTKs receptor tyrosine kinases
  • integrin-mediated adhesion alone can activate growth factor receptor phosphorylation in the absence of ligands (5) and integrins bind directly to growth factor receptors like EGFR and cMET (5,6).
  • Adhesion of the endothelial cells to extracellular matrix molecules also profoundly affects their angiogenetic properties. For instance, binding of endothelial cells to vitronectin by ⁇ v ⁇ 3 integrin positively regulates VEGFR2 activation via direct interaction between the two molecules. It has also been known for a long time that collagenous matrix can inhibit endothelial growth (7). However, the molecular nature of the matrix-dependent integrin- mediated signals that inhibit angiogenesis is not understood.
  • TCPTP T-cell protein tyrosine phosphatase
  • the present invention is based on a novel study where we surprisingly found that TCPTP is present in endothelial cells and that a ligation of a collagen-binding integrin or TCPTP activation could restrain VEGFR2 signalling and inhibit biological responses to VEGF.
  • TCPTP dephosphorylates and silences VEGFR2 and devised a peptide from the cytoplasmic domain of integrin ⁇ l to be used as a tool to inhibit VEGFR2 signalling via the activation of TCPTP.
  • the data obtained in our study indicate that endothelial cell growth during different steps of angiogenesis is regulated by microenvironmentally controlled phosphatase activities.
  • this invention concerns the use of an agent capable of activating T cell protein tyrosine phosphatase (TCPTP) in the manufacture of a pharmaceutical composition for use in a method for inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signalling in an individual.
  • TCP T cell protein tyrosine phosphatase
  • the invention concerns the use of an agent capable of activating T cell protein tyrosine phosphatase (TCPTP) in the manufacture of a pharmaceutical composition for use in a method for inhibiting angiogenesis or for treatment or prevention of a disease benefiting from suppression of the growth of blood vessels in an individual.
  • TCP T cell protein tyrosine phosphatase
  • the invention concerns the use of an agent capable of inhibiting T cell protein tyrosine phosphatase (TCPTP) in the manufacture of a pharmaceutical composition for use in a method for stimulating angiogenesis or for treatment or prevention of a disease benefiting from increasing the growth of blood vessels in an individual.
  • TCP T cell protein tyrosine phosphatase
  • the invention concerns a peptide comprising the amino acid sequence RPLKKKMEK (SEQ ID NO 1), wherein all the amino acids are D- amino acids, or a vector being capable of expressing said peptide in a mammalian cell.
  • this invention concerns a method for inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signalling in an individual, by administering to said individual an effective amount of an agent capable of activating T cell protein tyrosine phosphatase (TCPTP).
  • VEGFR2 vascular endothelial growth factor receptor 2
  • the invention concerns a method for inhibiting angiogenesis or for treatment or prevention of a disease benefiting from suppression of the growth of blood vessels in an individual by administering to said individual an effective amount of an agent capable of activating T cell protein tyrosine phosphatase (TCPTP).
  • TCP T cell protein tyrosine phosphatase
  • the invention concerns a method for stimulating angiogenesis or for treatment or prevention of a disease benefiting from increasing the growth of blood vessels in an individual by administering to said individual an effective amount of an agent capable of inhibiting T cell protein tyrosine phosphatase (TCPTP).
  • TCP T cell protein tyrosine phosphatase
  • this invention concerns an agent capable of activating T cell protein tyrosine phosphatase (TCPTP) for use in a method for inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signalling in an individual.
  • TCP T cell protein tyrosine phosphatase
  • VEGFR2 vascular endothelial growth factor receptor 2
  • the invention concerns an agent capable of activating T cell protein tyrosine phosphatase (TCPTP) for use in a method for inhibiting angiogenesis or for treatment or prevention of a disease benefiting from suppression of the growth of blood vessels in an individual.
  • TCP T cell protein tyrosine phosphatase
  • the invention concerns an agent capable of inhibiting T cell protein tyrosine phosphatase (TCPTP) for use in a method for stimulating angiogenesis or for treatment or prevention of a disease benefiting from increasing the growth of blood vessels in an individual.
  • TCP T cell protein tyrosine phosphatase
  • FIG. 1 Adhesion of HUVEC to collagen activates TCPTP.
  • A Immunoblotting of ⁇ l and ⁇ l integrins, TCPTP and VEGFRs from HUVEC lysates (HeLa lysates and tubulin blots are shown as controls)
  • TCPTP binds to VEGFR2, but not to VEGFRl, and dephosphorylates it in a phospho-site specific manner.
  • A Microscopy images from two-color immunofluorescence stainings show co-localization of integrin ⁇ l ⁇ l with TCPTP and VEGFR2 (arrows) in HUVEC adhering to collagen. Bar, 10 ⁇ m.
  • B HEK293 cells transiently transfected with VEGFR2 and substrate-trapping mutant of TCPTP (TCPTP-D 182A) were treated with VEGF (100 ng/ml, 15 min) and immunoprecipitated (IP) with anti-TCPTP or control (IgG) antibodies and blotted as indicated.
  • HEK293 cells were transfected with VEGFR2, treated with VEGF and immunoprecipitated (IP) with anti-VEGFR2. Immunoprecipitates were incubated in the presence of recombinant TCPTP or buffer control, and immunoblotted using phospho-specific VEGFR2 antibodies. Total VEGFR2 was blotted as a control. Representative experiments out of 3 with similar results are shown.
  • FIG. 3 TCPTP activity controls VEGF-dependent responses in endothelial cells.
  • HUVEC were transfected with the constitutively active TCPTP (TC37) or vector control (pCG), stimulated or not with VEGF, and stained for VEGFR2 (red) and nuclei (blue) after fixation and permeabilization. Arrows indicate VEGFR2 vesicles.
  • (D) HUVEC were transfected as in (A) and chemotaxis towards VEGF was measured using a Trans well assay for 4 hours. Cells adhering to the bottom of the filter were fixed and stained with crystal violet. Number of migrated cells was counted (mean ⁇ SEM, n 3).
  • FIG. 4 ⁇ l integrin activates TCPTP and attenuates VEGFR2 signalling.
  • ⁇ lTAT ⁇ l integrin cytoplasmic tail fusion peptide
  • ScrTAT TAT-scramble control fusion peptide
  • (B) HUVEC were treated as in (A) and VEGFR2 kinase activity (mean ⁇ SD, n 3) was measured from the cell lysates. **, p ⁇ 0.01.
  • (C) Recombinant, purified TCPTP was incubated with ⁇ l or ⁇ 2 cytoplasmic tail peptides (L-amino acids) or with ⁇ l and scrambled TAT peptides (D-amino acids) and analyzed for phosphatase activity (mean ⁇ SD, n 3).
  • FIG. 5 Controlled TCPTP activation inhibits VEGF-driven capillary formation, chemokinesis and chemotaxis.
  • HUVEC were cultured in fibrin gels and treated with or without VEGF and TAT peptides (200 nM). After 24 h the formation of capillary tubes was semiquantitatively scored.
  • B HUVEC were plated on gelatin in the presence of the peptides (200 nM) or left untreated for 1 h. The cells were stimulated as indicated and migration of individual cells was tracked using time- lapse microscopy. Cumulative migration distance was plotted for randomly picked individual cells.
  • HUVEC were treated with 200 nM peptides and the transmigration assay was performed as in Fig 3D.
  • D HUVEC were transfected with siRNA against TCPTP or a control siRNA, and TCPTP expression was analysed with immunofluorescence. Equal exposures of TCPTP (green) and DAPI (blue) stainings are shown. 48 h post-transfection cells were subjected to transmigration assay as in (D).
  • PAE cells stably expressing VEGFRl were transfected with the substrate-trapping mutant of TCPTP (TCPTP-D 182A), treated with VEGF (100 ng/ml, 15 min) or left untreated and immunoprecipitated (IP) with anti-TCPTP or control (IgG) antibodies and blotted as indicated. Aliquots of the lysates were also analysed to control for equal protein expression.
  • FIG. 7 TCPTP activation through the cytoplasmic domain of ⁇ l integrin inhibits internalization of VEGFR2.
  • ⁇ lTAT ⁇ l integrin cytoplasmic tail fusion peptide
  • ScrTAT TAT-scramble control fusion peptide
  • HUVEC were treated as in (A) and subjected to trypsin digestion on ice.
  • VEGFR2 protected from trypsin was detected by immunoblotting.
  • the intensity of VEGFR2 bands was quantified using densitometry and expressed as arbitrary units. A representative experiment out of 3 with similar results is shown.
  • FIG. 8 VEGF-induced capillary formation is attenuated by ⁇ l -integrin. HUVEC were plated on gelatin, overlayed by collagen and treated with or without VEGF and TAT peptides (200 nM). After 24 h the number of closed polygons formed by endothelial tubes was enumerated microscopically. The mean scores from a representative assay are shown.
  • FIG. 10 Activation of TCPTP does not inhibit VEGF-responses in confluent HUVEC.
  • Serum-starved HUVEC were plated to confluency on gelatin and treated for 1 h with ⁇ l integrin cytoplasmic tail fusion peptide ( ⁇ lTAT) or TAT- scramble control fusion peptide (ScrTAT) (both at 200 nM) or not treated, followed by VEGF- induction (15 min) when indicated.
  • Cell lysates were immunoblotted for phosphorylated VEGFR2 (or tubulin as a control).
  • (B) Confluent HUVEC on gelatin were treated with VEGF in the presence or absence of ⁇ lTAT or ScrTAT peptide and the number of live cells was detected using WST-I reagent (mean ⁇ SD, n 3).
  • Figure 11 shows the mRNA sequence (SEQ ID NO 2) of TCPTP where the binding sites for two siRNA:s are marked.
  • treatment shall be understood to include complete curing of a disease or disorder, as well as amelioration or alleviation of said disease or disorder.
  • prevention shall be understood to include complete prevention, prophylaxis, as well as lowering the individual's risk of falling ill with said disease or disorder.
  • the term "individual” refers to a human or animal subject.
  • an effective amount is meant to include any amount of an agent according to the present invention that is sufficient to bring about a desired therapeutical result, especially upon administration to an animal or human subject.
  • inhibitorting or “inhibition” shall be understood to include not only complete inhibition but also any grade of suppression.
  • peptide shall be understood to include peptides of L-amino acids, D- amino acids or both unless the form of the amino acids is explicitly expressed.
  • the agent to be used for activating of TCPTP in order to inhibit VEGRF2 signalling can either be a small molecule able to activate TCPTP, or a peptide comprising the amino acid sequence RPLKKKMEK (SEQ ID NO 1). This sequence is specific for the cytoplasmic tail of alpha- 1-integrin.
  • the peptide can, according to one embodiment, be exactly the amino acid sequence RPLKKKMEK (SEQ ID NO 1), which could be administered to the cell by microinjection, for example.
  • the peptide can be a longer chain encompassing the amino acid sequence RPLKKKMEK (SEQ ID NO 1).
  • the peptide (the chain RPLKKKMEK (SEQ ID NO 1) or a longer chain encompassing the same) can be part of a cell membrane permeable fusion protein.
  • a fusion protein can be mentioned a fusion protein comprising a protein transduction domain of HIV transcriptional transactivator (TAT) protein and the amino acid sequence RPLKKKMEK (SEQ ID NO 1).
  • TAT protein transduction domain of HIV transcriptional transactivator
  • YGRKKRRQRRRWKLGFFKRPLKKKMEK SEQ ID NO 3
  • the sequence YGRKKRRQRRR (SEQ ID NO 4) is derived from TAT and the sequence WKLGFFK (SEQ ID NO 5) is derived from the integrin (typical for any alpha- integrin).
  • a suitable cell membrane permeable fusion protein can be mentioned a fusion protein of the antennapedia peptide RQIKIWFQNRRMKWKK (SEQ ID NO 6) and the amino acid sequence RPLKKKMEK (SEQ ID NO 1).
  • the sequence of such a fusion protein is RQIKIWFQNRRMKWKKWKLGFFKRPLKKKMEK (SEQ ID NO 7).
  • all the amino acids in the RPLKKKMEK are D-amino acids.
  • the peptide RPLKKKMEK (SEQ ID NO 1) is a part of a fusion protein, preferably all amino acids of such a fusion protein are D-amino acids.
  • the agent to be administered can be a vector, comprising a nucleic acid being capable of expressing the desired peptide in a mammalian cell.
  • the nucleic acid can be inserted in a DNA sequence, an RNA sequence or in a viral vector.
  • a viral vector is typically based on an adenovirus, an alphavirus, adeno-associated virus, a retrovirus or a herpes virus.
  • a viral vector is typically based on an adenovirus, an alphavirus, adeno-associated virus, a retrovirus or a herpes virus.
  • suitable small molecules for activating TCPTP can be mentioned Ruthenium Red,spermidine, Mitoxantrone and MDL-26,630 trihydrochloride.
  • Inhibiting VEGFR2 signalling by administering an effective amount of an agent capable of activating TCPTP is useful for inhibiting angiogenesis or for treatment or prevention of a disease benefiting from suppression of the growth of blood vessels in an individual.
  • diseases that could be treated or prevented by this method can be mentioned retinopathy, age related macular degeneration, hemangioma, arthritis, psoriasis, atherosclerosis and cancer. This method is very effective in destroying the vasculature of tumors.
  • the peptide comprising the sequence RPLKKKMEK (SEQ ID NO 1) is preferably brought in a form which allows permeation through the cell membrane.
  • a peptide can be admixed with any carrier that is suitable for parenteral administration of the composition.
  • the peptide can be complexed with a lipid, packed in a liposome, incorporated in a cyclodextrin or other complexing agent, a bioresorbable polymer or other suitable carrier for controlled release administration, or encompassed in a a nanoparticle or hydrogel.
  • an expression vector encompassing a nucleic acid encoding a peptide comprising the amino acid sequence RPLKKKMEK (SEQ ID NO 1) can be administered to the individual.
  • the nucleic acid can encode the exact sequence
  • the vector can, for example, be complexed with a lipid, packed in a liposome, incorporated in a cyclodextrin or other complexing agent, a bioresorbable polymer or other suitable carrier for controlled release administration, or encompassed in a a nanoparticle or hydrogel.
  • the therapeutically effective amount of the peptide or expression vector to be given to a patient in need of such treatment may depend upon a number of factors including, for example, the age and weight of the patient, the precise condition requiring treatment and its severity, and the route of administration. The precise amount will ultimately be at the discretion of the attending physician.
  • practice of the present invention may involve any dose, combination with other therapeutically effective drugs, pharmaceutical formulation or delivery system for parenteral administration.
  • the peptide or expression vector can be administered systemically or locally.
  • suitable routes of administration can be mentioned intravenous, intramuscular, subcutaneous injection, inhalation, topical, ocular, sublingual, nasal, rectal, intraperitoneal delivery and iontophoresis or other transdermal delivery systems.
  • VEGFR2 signalling and consequently angiogenesis
  • an agent capable of inhibiting TCPTP can be stimulated in an individual by administering an effective amount of an agent capable of inhibiting TCPTP.
  • This method is useful for treatment or prevention of a disease benefiting from increasing the growth of blood vessels in an individual.
  • diseases that could be treated or prevented by this method can be mentioned coronary artery disease, peripheral arterial occlusion or another disease benefiting from promoting of neo vascularis ation.
  • agents for inhibiting TCPTP can be mentioned i) a small inhibitory RNA (siRNA) oligonucleotide capable of inhibiting TCPTP expression ii) a vector being capable of expressing said inhibitory RNA as a hairpin RNA (shRNA) in mammalian cell, or iii) a small molecule able of inhibiting TCPTP.
  • siRNA small inhibitory RNA
  • shRNA hairpin RNA
  • siRNA has become important in the development of new therapies in the last years. O Heidenreich presents an overview of pharmaceutical applications in the article "Forging therapeutics from small interfering RNAs in European Pharmaceutical Review Issue 1, 2005. The principle has particularly been suggested for the treatment of tumors and carcinomas, sarcomas, hypercholesterolemia, neuroblastoma and herpetic stromal keratitis.
  • siRNA duplex molecule comprises an antisense region and a sense strand wherein said antisense strand comprises sequence complementary to a target region in an mRNA sequence encoding a certain protein, and the sense strand comprises sequence complementary to the said antisense strand.
  • the siRNA duplex molecule is assembled from two nucleic acid fragments wherein one fragment comprises the antisense strand and the second fragment comprises the sense strand of said siRNA molecule.
  • the sense strand and antisense strand can be covalently connected via a linker molecule, which can be a polynucleotide linker or a non- nucleotide linker.
  • the length of the antisense and sense strands are typically about 19 to 21 nucleotides each.
  • the antisense strand and the sense strand both comprise a 3'-terminal overhang of a few, typically 2 nucleotides.
  • the 5'-terminal of the antisense is typically a phosphate group (P).
  • the siRNA duplexes having terminal phosphate groups (P) are easier to administrate into the cell than a single stranded antisense.
  • RISC RNA-induced silencing complex
  • complementary means that the nucleotide sequence can form hydrogen bonds with the target RNA sequence by Watson-Crick or other base-pair interactions.
  • the term shall be understood to cover also sequences which are not 100 % complementary. It is believed that also lower complementarity might work. However, 100 % complementarity is preferred.
  • the siRNA shall, when used as a pharmaceutical, be introduced in a target cell.
  • the delivery can be accomplished in two principally different ways: 1) exogenous delivery of the oligonucleotide or 2) endogenous transcription of a DNA sequence encoding the oligonucleotide, where the DNA sequence is located in a vector.
  • RNA normal, unmodified RNA has low stability under physiological conditions because of its degradation by ribonuclease enzymes present in the living cell. If the oligonucleotide shall be administered exogenously, it is highly desirable to modify the molecule according to known methods so as to enhance its stability against chemical and enzymatic degradation. Modifications of nucleotides to be administered exogenously in vivo are extensively described in the art. Principally, any part of the nucleotide, i.e the ribose sugar, the base and/or internucleotidic phosphodiester strands can be modified.
  • the internucleotidic phosphodiester linkage can, for example, be modified so that one ore more oxygen is replaced by sulfur, amino, alkyl or alkoxy groups.
  • the base in the nucleotides can be modified.
  • the oligonucleotide comprises modifications of one or more 2'-hydroxyl groups at ribose sugars, and/or modifications in one or more internucleotidic phosphodiester linkages, and/or one or more locked nucleic acid (LNA) modification between the T- and 4'-position of the ribose sugars.
  • LNA locked nucleic acid
  • Particularly preferable modifications are, for example, replacement of one or more of the 2'-OH groups by 2'-deoxy, 2'-O- methyl, 2'-halo, eg. fluoro or 2'-methoxyethyl.
  • oligonucleotides where some of the internucleotide phoshodiester linkages also are modified, e.g. replaced by phosphorothioate linkages.
  • siRNA examples of useful siRNA:s and vectors are disclosed in reference 8, paragraph Transfections. Two different annealed siRNAs targeting TCPTP (ggcacaaaggaguuacauctt and ggaguuacaucuuaacacatt) were disclosed. These sequences (except for the overhangs tt) represent the sense strands of the duplexes, which also comprise antisense strands which complementary to the sense strands.
  • the target sites for these siRNA:s are indicated as underlined or bold, respectively, in Figure 11. However, also other useful target regions at the target RNA can be used. A useful target region can easily be identified by using any of the numerous academic or commercially affiliated algorithms that have been developed to assist scientists to locate utilizable siRNA sequences. As examples of such software systems can be mentioned siDirect (http://design.RNAi.jp/) (Nucleic Acids Res. 2004 JuI 1;32: W 124-9); TROD (T7 RNAi Oligo Designer
  • TCPTP shRNA construct was made by cloning the sequence corresponding to the functional TCPTPl oligo into pRNA-U6.1/Neo (Genscript, Piscataway, NJ) according to the manufacturer's instructions.
  • TCPTP T-cell protein tyrosine phosphatase
  • TCPTP can also be activated in endothelial cells using cell-permeable peptides from the cytoplasmic tail of the collagen-binding integrin ⁇ l.
  • This controlled activation of TCPTP results in inhibition of VEGF- triggered endothelial cell proliferation, chemokinesis, chemotaxis, and capillary formation.
  • TCPTP-dependent inhibition of VEGF signalling specifically targets actively growing endothelial cells.
  • Antibodies, plasmids and reagents Antibodies, plasmids and reagents. Antibodies against TCPTP (mAb CF4, Calbiochem and polyclonal Ab, BD Pharmingen), VEGFRl (Santa Cruz), total VEGFR2 (55B11), and phosphotyrosines 996, 1175 and 1214 of VEGFR2 (all from Cell Signaling Technology), ⁇ l-integrin (mAb 1973 (clone FB12) and Abl934, Chemicon), ⁇ l-integrin (mAb 2252, Chemicon), tubulin (Hybridoma Bank), and control mouse IgG were used. HRP- and Alexa conjugated second-stage reagents were used, as appropriate.
  • a plasmid containing a 37 kDa constitutively active form of TCPTP (TC37 in pcDNA3.1(+)), a substrate-trapping mutant of TCPTP (pCG- TC45-D182A), the control pCG-vector and VEGFR2 expression plasmid have been described (24,25).
  • siRNA against TCPTP and a control siRNA (8) were from Ambion.
  • Peptides containing the cytoplasmic tails of ⁇ l or ⁇ 2 integrins, the ⁇ l-tail, the sequence of which is RPLKKKMEK (SEQ ID NO 1), fused to the 11 amino acid long TAT peptide (the sequence of which is YGRKKRRQRRR, (SEQ ID NO 4)) and scrambled TAT peptides (8) were synthesized by Innovagen.
  • ⁇ lTAT and ScrTAT peptides were also synthesized with D-amino acids.
  • ⁇ lTAT YGRKKRRQRRRWKLGFFKRPLKKKMEK (SEQ ID NO 3) (where YGRKKRRQRRR (SEQ ID NO 4) is the 11 aa long TAT peptide, RPLKKKMEK (SEQ ID NO 1) is the ⁇ l-tail of the integrin and WKLGFFK (SEQ ID NO 5) is derived from the integrin); and Scr-TAT: YGRKKRRQRRRLKGWRFKLKPKFKEMK (SEQ ID NO 8).
  • HUVEC Endothelial cell growth medium
  • HUVEC were plated on type IV collagen, fixed with 4% paraformaldehyde and VEGFR2, TCPTP and ⁇ l integrin were visualized using indirect immunofluorescence stainings.
  • the samples were mounted in Vectashield with DAPI (Vector Laboratories, CA) and analyzed using confocal microscope (Axioplan 2 with LSM 510; Carl Zeiss Microimaging, Inc.).
  • the plasmid for substrate-trapping mutant of TCPTP was cotransfected with a plasmid encoding VEGFR2 into HEK293 cells or transfected into PAE-Fltl cells using Lipofectamine 2000. After 1 d the cells were serum- starved (overnight), stimulated with VEGF (50 ng/ml) for 10 min or left untreated, washed with ice-cold PBS and lysed. Postcentrifugation lysates were precleared with Protein-G-sepharose beads and subjected to immunoprecipitation with anti- TCPTP or control mouse IgG. After washings, the immunocomplexes were resolved on SDS-PAGE and subjected to immunoblotting. Aliquots of the original lysates (5% from IP input) were analyzed in parallel to control for protein expression.
  • Phosphatase and kinase assays were performed as described (8). Briefly, HUVEC were plated to collagen or gelatin coated plates and allowed to adhere for 1 h. TCPTP, SHP2 and control immunocomplexes were isolated from cell lysates, resuspended in the phosphatase assay buffer and assayed for phosphatase activity using 6,8-difluoro-4-methylumbelliferyl phosphate; Molecular Probes) as a substrate in the presence of a serine / threonine phosphatase inhibitor cocktail (Sigma). Thereafter, the antigens from the beads were used for immunoblotting. The phosphatase assays with purified TCPTP protein were performed as described (8) using diFMUP as a substrate.
  • VEGFR2 kinase activity was studied using Kinase-Glo Plus kinase assay kit according to manufacturer's protocol (Promega) with 25 ⁇ M ATP. Briefly, HUVEC plated on gelatin were serum- starved overnight, treated with 400 nM ⁇ lTAT or ScrTAT or left untreated and stimulated with 100 ng/ml VEGF for 15 min, when indicated. The cells were lysed to ice-cold lysis buffer (1% NP-40, 20 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol, 2.5 mM EDTA, complete protease inhibitor (Roche) and phosphatase inhibitor coctails I and II (Sigma)).
  • Kinase-Glo Plus kinase assay kit according to manufacturer's protocol (Promega) with 25 ⁇ M ATP. Briefly, HUVEC plated on gelatin were serum- starved overnight, treated with 400 nM
  • the lysates were incubated in anti-VEGFR2 or control IgG-coated wells with shaking for 2 h and washed 3 times with lysis buffer and 3 times with cold kinase assay buffer (20 mM Hepes pH 8, 0.1 mM Na 3 VO 4 , 10 mM MnCl 2 , 1 mM DTT). Kinase assay was performed for 10 min at +37°C.
  • VEGFR2 was immunoprecipitated from the lysates (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 2.5 mM EDTA, 1% NP-40, Complete protease inhibitor cocktail, Sigma PPASE inhibitor coctails I and II).
  • the beads were washed with the phosphatase assay buffer (10 mM Hepes, 150 mM NaCl, 1 mM DTT), and exposed to recombinant TCPTP (0,12 mg/ml) or buffer only for 10 min at +37°C. Immunoblotting was used to detect total VEGFR2 and specific phosphotyrosines.
  • HUVEC proliferation and survival assays The number of viable HUVEC was analyzed with WST-I reagent (Roche). Cell proliferation was measured with BrdU assay (Cell proliferation ELISA Biotrak system, Amersham) from siRNA- transfected HUVEC grown in RPMIl 640 + 1% FBS + 2 mM L-glutamine supplemented with 80 ng/ml VEGF.
  • Tube formation assays In the fibrin-gel in vitro angiogenesis assay (Chemicon) HUVEC were plated onto a fibrin-gel, and overlaid with a second fibrin-gel after an overnight culture. In the collagen overlay assay (26) HUVEC were plated on gelatin-coated wells and allowed to attach. Then a collagen gel (0.2 M Hepes, 1OxDMEM and Vitrogen 100 purified collagen I) was added. In both assays, after the polymerization of the top gel the endothelial cell growth medium with or without VEGF (50 ng/ml) was added in the presence or absence of ⁇ lTAT or scrambled TAT peptides (400 nM).
  • the formation of cellular networks was quantified from three randomly-selected fields from each well.
  • fibrin gel assay scores 0-5 were assigned to each field based on cellular aligning, capillary tube formation, sprouting, and formation of polygons according to the manufacturer's instructions.
  • collagen overlay assay the number of closed polygons formed by capillary tubes was quantified.
  • HUVEC were transfected with control plasmid or with constitutively active 37 kDa TCPTP, grown on gelatin and stimulated with
  • VEGFR2 internalization was followed microscopically (see above).
  • cells were placed on ice and treated with cold trypsin (1 mg/ml, 30 min), which cleaves cell-surface expressed VEGFR2, but leaves internalized receptor intact (12).
  • the amount of VEGFR2 in each sample was analyzed using immunoblotting.
  • Time-lapse chemokinesis assays HUVEC were plated on gelatin-coated wells and allowed to attach for 1 h in the presence or absence of 200 nM ⁇ lTAT or ScrTAT and 20 ng/ml VEGF.
  • Migration of individual cells was recorded at 5 min intervals for 4 h using 2Ox objective and digital video recording. The migration pathway of at least ten randomly picked cells was tracked using the Metamorph program. The cumulative migration distances were also plotted.
  • HUVEC electroporated with the DNA or siRNA or non- transfected HUVEC were treated with 200 nM ⁇ lTAT, ScrTAT or left untreated for I h.
  • Transwell filters (8 ⁇ m pore size, Costar, Cambridge, MA) were coated with gelatin and medium (RPMI, 4 mM L-glutamine, 10% FBS) and VEGF (100 ng/ml) were added to the lower chamber. Cells were harvested from the culture plates, and transferred into the Transwell inserts (4x10 cells/well) in RPMI with 0.2% soybean trypsin inhibitor. Chemotaxis was allowed to proceed for 4 h (+37°C, 5% CO 2 ).
  • the inserts were removed, and the non-migrated cells from the top of the insert were removed by scraping with a cotton swab.
  • the inserts were fixed in 4% paraformaldehyde, cells stained with 0.5% crystal violet and the number of migrated cells was counted using a microscope.
  • TCPTP Adhesion of endothelial cells to collagen activates TCPTP.
  • human endothelial cells could express TCPTP and that adhesion to collagen via ⁇ l integrin might affect its activity.
  • Immunoblotting experiments revealed that TCPTP protein and ⁇ l ⁇ l integrin are present in early-passage human umbilical vein endothelial cells (HUVEC; Fig IA). FACS-analyses further revealed that ⁇ l integrin is expressed on the surface of HUVEC (not shown).
  • TCPTP binds VEGFR2 but not VEGFRl.
  • ⁇ l integrin TCPTP and VEGFR could function as a signalosome we first determined their localization in endothelial cells. Immunofluorescence analyses showed that ⁇ l ⁇ l colocalizes with TCPTP in HUVEC adhering to collagen (Fig 2A). Interestingly, VEGFR2 also showed partial colocalization with ⁇ l integrin in these cells (Fig 2A).
  • a substrate-trapping technique Binding of a substrate to TCPTP-D 182A mutant results in a stable interaction between the enzyme and the substrate (9).
  • VEGFR2 protein was readily co-immunoprecipitated with TCPTP-D 182A (Fig 2B).
  • TCPTP-D 182A did not associate with VEGFRl in VEGF-treated cells overexpressing the receptor (Fig 6).
  • VEGFR2 is a substrate for TCPTP in vivo.
  • TCPTP dephosphorylates VEGFR2 in a site-specific manner.
  • VEGFR2 contains several critical tyrosine residues that are autophosphorylated or bind distinct effector molecules. Some of the most important ones are Tyr996 which is one of the autophosphorylation sites, Tyrl 175 which binds to PLC ⁇ and Shb, and Tyr 1214 which triggers p38 cascade through unknown intermediates (3). Phosphorylation assays showed that purified, recombinant TCPTP was able to dephosphorylate isolated VEGFR2 in vitro (Fig 2C).
  • TCPTP showed a striking specificity to two tyrosine residues studied (Y1214 and Y996), while tyrosine 1175 was not a TCPTP target.
  • TCPTP binds to VEGFR2 both in vivo and in vitro and it dephosphorylates the receptor in a phosphosite-specific manner.
  • TCPTP controls internalization of VEGFR2.
  • VEGFR2 is internalized upon engagement with VEGF and recent data suggests that internalized receptor is actively signalling (10,11). This prompted us to investigate whether TCPTP activity controls the internalization.
  • VEGF treatment resulted in efficient endocytosis of VEGFR2 to early endosome -resembling vesicles in sparse mock-transfected HUVEC cells (Fig 3A).
  • Transfection of constitutively active 37 kDa TCPTP into HUVEC clearly inhibited VEGFR2 internalization to endosomes.
  • TCPTP activity regulates responsiveness of endothelial cells to VEGF.
  • siRNA siRNA-induced proliferation of HUVEC was 28 ⁇ 6% higher than in controls (Fig 3C), which is consistent with the ability of TCPTP to dephosphorylated VEGFR2 in the absence of TCPTP.
  • a constitutively active TCPTP was then used in chemotaxis assays to analyze the effect of enhanced VEGFR2 dephosphorylation on VEGF-driven cell migration. We found that VEGF stimulated chemotaxis of vector-transfected HUVEC by >2-fold.
  • TCPTP constitutively active TCPTP
  • Fig 3D VEGF-driven responses in HUVEC.
  • the cytoplasmic domain of ocl integrin activates TCPTP and inhibits VEGFR- 2 activity.
  • a cell-permeable TAT-fusion peptide from the cytoplasmic tail of integrin ocl but not from that of ⁇ 2, ⁇ 5, ⁇ lO or al l integrals, specifically activates TCPTP (8). Therefore, we studied whether this peptide could be used as a tool to trigger TCPTP-dependent VEGFR2 dephosphorylation in normal endothelial cells.
  • ⁇ lTAT peptide effectively attenuated this TAT-mediated induction of VEGFR2 phosphorylation as well (Fig 4A).
  • VEGFR2 kinase activities from HUVEC.
  • VEGF-stimulation the kinase activity increased about 10-fold in cells treated with no peptide or with the scrambled control TAT peptide.
  • ⁇ lTAT peptide completely abolished the kinase activity of VEGFR2 in VEGF- treated HUVEC (Fig 4B).
  • the ⁇ lTAT peptide can be used to dephosphorylate and inactivate VEGFR2 in HUVEC.
  • TAT-fusion peptides To increase the half-life of TAT-fusion peptides in cells, we designed novel TAT peptides with D-amino acids instead of normal L-isomers.
  • ⁇ l -D-TAT was found to be a very good activator of TCPTP. Again the specificity of the TCPTP activation was evident, inasmuch as the scrambled TAT-fusion made of D-isomers had no effect (Fig 4C).
  • the ⁇ l -D-TAT specifically inhibited internalization of VEGFR2 in HUVEC ( Figure 7), which was consistent with the effects seen with constitutively active TCPTP.
  • Controlled TCPTP activation can be exploited to inhibit endothelial proliferation, migration and capillary morphogenesis.
  • Capillary morphogenesis requires adhesion, migration, proliferation, and differentiation of endothelial cells and VEGFR2 plays a major role in all of these steps (2).
  • ⁇ lTAT peptide inhibited the capillary tube formation by 60% when compared to non-treated cells and by 40% when compared to control peptide treated cells in a fibrin gel assay (Fig 5A). Similar inhibition of capillary formation by ⁇ lTAT peptide was found in a collagen overlay assay ( Figure 8). Together these results show that activation of TCPTP through the ⁇ l cytoplasmic domain leads to inhibition of VEGF-driven capillary formation.
  • ⁇ lTAT treatment inhibited VEGF-induced chemokinesis by 76+4%, while ScrTAT had no significant effect. It was also clear from the images that neither TAT peptide was harmful to the cells, since all cells were viable and entered occasionally mitosis (not shown). VEGF-induced chemotaxis of HUVEC was also inhibited by treatment with ⁇ lTAT peptide, but not with the scrambled TAT peptide (Fig 5C).
  • VEGF-induced HUVEC migration is mediated via TCPTP.
  • ⁇ lTAT had no inhibitory effects on the VEGF induced chemotaxis of TCPTP siRNA-treated cells, whereas ⁇ lTAT effectively inhibited VEGF-induced migration of scrambled siRNA treated cells.
  • TCPTP is the target of the cytoplasmic peptide of ⁇ l integrin in HUVEC and that decreased TCPTP activity results in augmented VEGFR2-mediated proliferation and migration of endothelial cells.
  • TCPTP activation selectively targets actively growing endothelial cells.
  • An ideal anti-angiogenetic compound would spare normal confluent endothelial cells in mature vessels and only target newly forming vessels.
  • a cell membrane-permeable peptide from ⁇ l -integrin efficiently attenuates VEGFR2-driven functions such as proliferation, migration, and tube formation in actively growing cells.
  • VEGF upregulates expression of ⁇ l ⁇ l and ⁇ 2 ⁇ l integrins in microvascular endothelial cells (14).
  • a combination of function-blocking ⁇ l and ⁇ 2 antibodies inhibit VEGF-driven angiogenesis in mouse skin (14) and in tumor xenografts (15).
  • cross-linking of ⁇ l integrin with the exactly same function-blocking monoclonal antibody leads to activation of TCPTP in malignant epithelial cells (8). Therefore, the inhibitory effects of anti- ⁇ l antibodies on vascular growth in vivo may be due to antibody-triggered activation of TCPTP in addition to blocking ⁇ l ⁇ l interaction with collagen.
  • Anti-VEGF antibodies are in clinical use and VEGFR2 kinase inhibitors are being developed for blocking neovascularization in several diseases (2).
  • Our data suggest that inhibition of VEGFR2 activity by inducing TCPTP would be a novel strategy for antagonizing VEGF-activity. Since TCPTP-dependent VEGFR2 inactivation was only seen in subconfluent but not in confluent cells, its modulation might spare the normal vessels. Since TCPTP activation also directly inhibits the growth of epithelial cancer cells in vitro, it might simultaneously target both the malignant cells and the vessels nourishing them. To obtain suitable tools to test this hypothesis in vivo we have started to screen for novel small molecule TCPTP activators.
  • VEGFR2 Upon VEGF stimulation, VEGFR2 is rapidly internalized and the rate of internalization is governed by phosphorylation (10,16). In contrary to the earlier dogma, internalized cell surface receptors maintain their signalling activity in the endosomes and even may recruit specific subset of down-stream effectors (17,18). In contact-inhibited cells, VEGFR2 is maintained inactive and prevented from being internalized by the VE-cadherin- ⁇ -catenin complex targeting VEGFR2 to the vicinity of density enhanced phosphatase- 1 (DEP-I), which dephosphorylates the receptor and prevents its internalization (11). Once internalized, the receptor is actively signalling and phosphorylated at least on tyrosines 1175 and 1214 (11).
  • DEP-I density enhanced phosphatase- 1
  • ⁇ l integrin is within a subclass of integrins that triggers Ras-pathway signalling via caveolin-1 (23). Therefore the cytoplasmic tail of ⁇ l integrin may effectively inhibit VEGFR2 internalization either by dephosphorylating VEGFR2 through induction of TCPTP or by spatial regulation of the ⁇ l ⁇ l-VEGFR2-caveolin complex at the cell surface.
  • ECM proteins have been shown to positively regulate VEGFR2 activity in few cases.
  • ligation of ⁇ v ⁇ 3 integrin supports VEGF-induced biological responses in endothelial cells in vitro and ⁇ v ⁇ 3 associates with VEGFR2 in response to VEGF treatment (19).
  • VEGF treatment 194204
  • ⁇ v ⁇ 3 associates with VEGFR2 in response to VEGF treatment (19).
  • VEGF treatment we defined the first molecular pathway that leads to matrix-guided inhibition of endothelial cell responsiveness to VEGF. This pathway is triggered by binding of endothelial cells to collagenous matrix through integrin ⁇ l, which leads to activation of TCPTP and inhibition of VEGFR2 signalling.

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Abstract

L'invention concerne un procédé pour inhiber la signalisation du récepteur 2 du facteur de croissance endothéliale vasculaire (VEGFR2) dans un sujet par l'administration d'une quantité efficace d'un agent capable d'activer la protéine tyrosine phosphatase des lymphocytes T (TCPTP), et un procédé pour inhiber l'angiogenèse et traiter ou prévenir des maladies apparentées à celle-ci. De plus, cette invention concerne un procédé pour stimuler l'angiogenèse ou pour le traitement ou la prévention d'une maladie bénéficiant de l'augmentation de la croissance de vaisseaux sanguins dans un sujet par l'inhibition de TCPTP.
PCT/FI2008/050262 2007-05-23 2008-05-13 Procédé pour inhiber ou stimuler l'angiogenèse dans un sujet WO2008142198A2 (fr)

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US10851073B2 (en) 2019-03-14 2020-12-01 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof
US10954202B2 (en) 2018-06-21 2021-03-23 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof

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US10954202B2 (en) 2018-06-21 2021-03-23 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof
US10851073B2 (en) 2019-03-14 2020-12-01 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof

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